Unraveling the impact of ZZZ3 on the mTOR/ribosome pathway in human embryonic stem cells homeostasis

Summary Embryonic stem cells (ESCs) are defined as stem cells with self-renewing and differentiation capabilities. These unique properties are tightly regulated and controlled by complex genetic and molecular mechanisms, whose understanding is essential for both basic and translational research. A large number of studies have mostly focused on understanding the molecular mechanisms governing pluripotency and differentiation of ESCs, while the regulation of proliferation has received comparably less attention. Here, we investigate the role of ZZZ3 (zinc finger ZZ-type containing 3) in human ESCs homeostasis. We found that knockdown of ZZZ3 negatively impacts ribosome biogenesis, translation, and mTOR signaling, leading to a significant reduction in cell proliferation. This process occurs without affecting pluripotency, suggesting that ZZZ3-depleted ESCs enter a “dormant-like” state and that proliferation and pluripotency can be uncoupled also in human ESCs.


Figure S1
. ZZZ3 is co-expressed with fibrillarin in the nucleolus and interacts with proteins involved in post-transcriptional processes and ribosome biogenesis.(Related to Fig. 1). A. GO for Cellular Components (A) and Molecular Functions (B) of differentially expressed genes selected on the basis of the p-values (p-value < 0.05 corrected by using Benjamini-Hochberg procedure), and fold-change (FC ≥ 2.5).GO analysis was performed in R using the Bioconductor package.C. Representative immunofluorescence images showing the colocalization of ZZZ3 (red) and Fibrillarin (FBL, green) were captured in wild-type ESC-1 and -2.Utilizing the JACoP BIOP colocalization plugin within the ImageJ software, the degree of colocalization was assessed by calculating the Pearson's correlation coefficient (r) across approximately 40 regions of interest (ROIs), representing the cell areas positive for FBL signal.These ROIs were randomly selected from three distinct stainings of both ESC lines.The intensity-based correlation analysis revealed positive values of the correlation coefficient, indicating the colocalization of ZZZ3 and FBL within the nucleolus of wild-type ESCs.D. Immunoprecipitation (IP) with an anti-ZZZ3 antibody followed by Western blot analysis was employed to validate interactome data.Specifically, interactions of ZZZ3 with DDX18, RPS6, and FBL are shown.Full-length uncropped blots are available in Supplementary File S1.

Figure S2. Knockdown strategies utilized to silence the ZZZ3 gene in two lines of human ESCs. A.
Schematic representation of the PiggyBac vector expressing shRNA against the human ZZZ3 gene under the control of the U6 promoter, utilized to establish stable ZZZ3 knockdown in hESCs.B. Immunoblot analysis was performed to evaluate the efficiency of ZZZ3 knockdown using three distinct shRNAs, labelled as KD 1 , KD 2 , and KD 3 .Notable, high efficient ZZZ3 knockdown was observed in cells transfected with shZZZ#2 (KD 2 ), which were subsequently utilized in this study.C and D. CRISPR interference (CRISPRi) targeting ZZZ3 mRNA and its validation via relative western blot analysis.CRISPRi-ZZZ3#1 and #3 exhibited effective ZZZ3 knockdown.E and F. TET-inducible shZZZ3 strategy and its validation by immunoblot analysis.The sequences of shRNA and guide RNA (gRNA) are provided in Supplementary Table S3.

Figure S3
. The expression of the three germ layers is not impaired upon ZZZ3 knockdown.(Related to Fig. 2). A. Immunofluorescence staining was performed to visualize the expression of specific markers indicative of ectoderm, endoderm, and mesoderm differentiation in the SCR control and ZZZ3 KD hESCs.Antibodies targeting key markers -such as OTX2 (ectoderm), BraT (mesoderm), and SOX17 (endoderm) -were used.Nuclei were counterstained with DAPI.Scale bar = 50 μm.Quantification of immunofluorescence images based on the percentage of positive cells was performed using ImageJ software.Data are presented as mean ± standard error of the mean (SEM) from n = 3 independent experiments.The difference observed between the SCR control and ZZZ3 KD hESCs was not statistically significant (ns) (graph on the right).B. Barplots show the gene expression levels of a panel of three germ layer regulators in SCR and ZZZ3 KD hESCs as determined by RNA-seq analysis.Mean expression levels ± SEM from three independent experiments is represented by bars, with individual data points overlaid as dots.Statistical analysis was performed using ANOVA, indicating non-significant differences (ns) between the experimental groups.C. Quantitative polymerase chain reaction (qPCR) was employed to measure the mRNA expression levels of the three germ layer markers on SCR and ZZZ3 KD EBs at day 10 of differentiation.Non-significant differences (ns) were detected between the experimental groups.D. Immunofluorescence staining of specific markers indicative of ectoderm (OTX2), endoderm (SOX17), and mesoderm (BraT) differentiation in the SCR control and ZZZ3 KD hESCs generated via CRISPRi system.Nuclei were counterstained with DAPI.Scale bar = 50 μm.Quantification of immunofluorescence images based on the percentage of positive cells was performed using ImageJ software.Data are presented as mean ± standard error of the mean (SEM) from n = 3 independent experiments.The difference observed between the SCR control and CRISPRi-ZZZ3sh hESCs was not statistically significant (ns) (graph on the right).3 and  5). A. Western blot analysis was conducted to assess the protein levels of cleaved caspase 9 and caspase 3 in SCR control and ZZZ3 KD hESCs (left); quantification of protein expression levels was performed using optical density (OD) measurement for each immunoblot shown.Data are presented as mean ± standard error of the mean (SEM) from n = 3 independent experiments.Significance was calculated vs. relative SCR ESCs using t-test.The difference observed between the groups was not statistically significant (ns) (right).B. Quantitative polymerase chain reaction (qPCR) was employed to measure the mRNA expression levels of BIM, BAX, and FAS genes in ZZZ3 KD vs. SCR control.Data are shown as mean ± SEM of three independent experiments and t-test was calculated vs. SCR cells (ns = not significant).C. Immunofluorescence staining for TUNEL was performed to detect DNA fragmentation indicative of apoptosis.Relative quantification was carried out to assess the extent of TUNEL-positive cells or fluorescence intensity.Scale bar: 50 µm.Quantification of immunofluorescence signals was performed using ImageJ software.Data are presented as mean ± standard error of the mean (SEM) from n = 3 independent experiments (at least 200 nuclei were analysed).Significance was calculated vs. relative SCR ESCs using t-test, ns = not significant.6). A. Western blot analysis was used to confirm the reduction of p-ERK 1/2, p-mTOR and p-AKT expression upon knockdown of ZZZ3 using SCR control and ZZZ3 KD hESCs generated via CRISPRi system.Quantification of protein expression levels was performed using optical density (OD) measurement.Data are presented as mean ± SEM from n = 3 independent experiments.Significance was calculated vs. relative SCR ESCs using t-test, * p ≤ 0.05, ** p ≤ 0.01.B-F.Immunoblot analysis was performed as readout of the rescue of key components of the PI3K/Akt/mTOR Signaling Pathways in ZZZ3 KD hESCs (ESC-1 and ESC-2 cell lines).B. and C. Representative immunoblots showing the protein expression levels of total ERK and phospho-ERK (Thr202/Tyr204) in ZZZ3 KD ESC-1 and ESC-2 treated and untreated with IGF-1 (100 mM, 30 minutes); D. and E. Representative immunoblots showing the protein expression levels of total mTOR and phospho-mTOR (Ser2448) in ZZZ3 KD hESCs treated and untreated with IGF-1; F. and G. Representative immunoblots showing the protein expression levels of total Akt and phospho-Akt (Ser473) in ZZZ3 KD hESCs treated and untreated with IGF-1.Quantification of immunoblot signals reveals a significant restoration of PI3K, Akt, and mTOR phosphorylation levels upon IGF-1 treatment in ZZZ3 knockdown hESCs compared to untreated knockdown cells.Data are presented as mean ± standard error of the mean (SEM) from n = 3 independent experiments.Significance was calculated vs. relative SCR ESCs cells using t-test, * p ≤ 0.05.

SUPPLEMENTARY TABLES
Table S3.Sequences used to induce ZZZ3 knockdown.Immunofluorescence staining.Immunofluorescence analysis was performed on Matrigel-coated glass coverslip in wells.Cells were fixed in 3.7% (vol/vol) formaldehyde for 15 minutes at room temperature, washed in PBS, permeabilized for 1h at RT in PBS + 0.3% Triton X-100 (Sigma-Aldrich) (PBST), and blocked for 1h at RT in PBST containing 10% of fetal bovine serum (FBS) (Thermo Fisher Scientific).Cells were subjected to immunostaining overnight at 4°C with primary antibodies (Table S4) diluted in blocking solution.

Figure S5 .
Figure S5.IGF-1 rescues dysregulated PI3K/Akt/mTOR signaling pathways in ZZZ3 knockdown hESCs.(Related to Fig.6). A. Western blot analysis was used to confirm the reduction of p-ERK 1/2, p-mTOR and p-AKT expression upon knockdown of ZZZ3 using SCR control and ZZZ3 KD hESCs generated via CRISPRi system.Quantification of protein expression levels was performed using optical density (OD) measurement.Data are presented as mean ± SEM from n = 3 independent experiments.Significance was calculated vs. relative SCR ESCs using t-test, * p ≤ 0.05, ** p ≤ 0.01.B-F.Immunoblot analysis was performed as readout of the rescue of key components of the PI3K/Akt/mTOR Signaling Pathways in ZZZ3 KD hESCs (ESC-1 and ESC-2 cell lines).B. and C. Representative immunoblots showing the protein expression levels of total ERK and phospho-ERK (Thr202/Tyr204) in ZZZ3 KD ESC-1 and ESC-2 treated and untreated with IGF-1 (100 mM, 30 minutes); D. and E. Representative immunoblots showing the protein expression levels of total mTOR and phospho-mTOR (Ser2448) in ZZZ3 KD hESCs treated and untreated with IGF-1; F. and G. Representative immunoblots showing the protein expression levels of total Akt and phospho-Akt (Ser473) in ZZZ3 KD hESCs treated and untreated with IGF-1.Quantification of immunoblot signals reveals a significant restoration of PI3K, Akt, and mTOR phosphorylation levels upon IGF-1 treatment in ZZZ3 knockdown hESCs compared to untreated knockdown cells.Data are presented as mean ± standard error of the mean (SEM) from n = 3 independent experiments.Significance was calculated vs. relative SCR ESCs cells using t-test, * p ≤ 0.05.

Table S5 . Primers used for quantitative PCR analysis.
, and incubated for 1 hour in darkness.ZZZ3 knockdown hESCs generated with piggyBac encoding for doxycycline-inducible ZZZ3 shRNA (DoxKD) were utilized to rescue the proliferation defect (Dox + /Dox -).Flow cytometry analysis was performed on the BD LSRFortessa x-20 Flow Cytometer, and data was processed using FlowJo software.TUNEL assay.For in vitro apoptosis detection, ESCs were initially seeded into matrigel-coated 8-well chamber slides.The cells were then fixed using 3.7% (vol/vol) formaldehyde (Sigma-Aldrich) and processed with the Click-iT™ Plus TUNEL Assay Kit AlexaFluor 594 (Thermo Fisher Scientific) following the manufacturer's instructions.After completing the assay, the cells were mounted with Dako Fluorescent Mounting Medium (Agilent), and images were captured using Leica microscopy systems (DMi8) equipped with Leica LAS X software(version 3.7.4.23463).TUNEL-positive cells were manually quantified using ImageJ software.